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Hydrolysis of polyesters by Nepenthes and Sarracenia pitcher fluids

In a first step, hydrolysis of polyesters films made from PET and from PBAT in the pitcher fluid of N. alata was studied based on the release of TPA. TPA concentration was found to increase in all samples consistently within the considered time frame: the highest TPA values corresponded to week 5, the final time point also due to the lifespan of the pitch (approximately 2 months). There was no overlapping and influence of TPA signal (retention time 5.7 min in the applied system) with tyrosine´s signal.

The highest concentration of TPA released from PBAT and PET of 1.61 mM and 1.27 mM, respectively, was recorded in Nepenthes alata for conditions in presence of alive mealworm (Tm_a) and JA (Fig. 2, Table S4). Interestingly, in the presence of JA and Tm_a around 10 times more TPA was released into the pitcher liquid compared to the respective polymer incubated alone in the pitcher (PET and PBAT, p < 0.001).

For PET, even addition of dry mealworms (Tm_d) led to a 3-fold increase of TPA (p < 0.01). However, for PBAT, this effect was not observed, as PBAT/Tm_d showed only a moderate increase compared to PBAT alone (p < 0.001). On the other hand, the presence of JA approximately doubled the release of TPA (0.66 mM vs. 0.37 mM, p < 0.001). In addition, the combined condition (JA + dry mealworm) already led to better depolymerization of PET, as TPA content in this set-up was significantly higher than the only-mealworm condition (p < 0.001). However, for PBAT, the combined condition (Tm_d/JA) did not result in a statistically significant increase in TPA content compared to the only-mealworm condition (Tm_d, p > 0.05). The samples with polyester only led to a comparatively low amount of TPA, however for the controls containing hormone only or mealworm only no TPA was detected. These results indicate that not only a mechanical stimulus (body of the mealworm), but also with the presence of the hormone JA could trigger the secretion of enzymes responsible for the hydrolysis of polyesters.

The polyesters were also hydrolyzed in S. purpurea pitcher fluids though to a lower extent when compared to N.alata (Fig. S1 and Table S5). Highest amounts of TPA were detected for PBAT (polymer only) and for PBAT/JA (polymer in presence of JA) for PBAT and PET/Tm_a/JA (alive mealworm and JA) for PET samples. Also the contribution of the hormone seemed to have a lower impact than in N. alata (12 times increase of TPA upon hormone introduction vs. only 2-fold in S. purpurea).

With the exception of PBAT/Tm_d and PET/Tm_d, all polymer-containing groups showed significant differences in TPA concentrations between N. alata and S. purpurea. Non-polymer-containing groups (Control, JA, Tm_a, Tm_a/JA, Tm_d) were not significantly different between the two plants. For the polymer-only conditions (PET and PBAT), S. purpurea released significantly more TPA than N. alata (p < 0.001). In contrast, all other polymer-containing conditions resulted in significantly higher TPA concentrations in N. alata compared to S. purpurea (p < 0.001).

In S. purpurea, the condition PET/Tm_a/JA consistently showed significantly higher TPA concentrations compared to PET, PET/JA, and PET/Tm_d (p < 0.05). For PBAT, both PBAT/Tm_a and PBAT/Tm_d had significantly lower TPA concentrations compared to PBAT/JA (p < 0.05). However, no significant differences were observed between most other conditions. These results suggest that while S. purpurea exhibits some variability in TPA release across conditions, its enzymatic activity is less pronounced compared to N. alata.

These findings provide a first evidence that pitcher fluids from N. alata and S. purpurea possess hydrolytic enzyme activity toward synthetic polyesters, with N. alata exhibiting significantly higher enzymatic efficiency. The hydrolysis of PET and PBAT, monitored via TPA release, was also enhanced by the presence of jasmonic acid (JA) and T. molitor (Tm), particularly in N. alata, where JA and alive Tm proved a synergistic effect. In these plants, the hormone has therefore an activating effect on the production of hydrolases, as the depolymerization was significantly higher when JA was present. In contrast, S. purpurea showed a more moderate response, but both plant digestive fluids showed a polyester degrading action, highlighting a previously underexplored natural system with potential relevance for plastic recycling.

As shown in previous studies⁷ hydrolysis of same size PET and PBAT films with 5 µM pure commercial enzyme (H.insolens cutinase) yielded 17 mM and 8 mM TPA, respectively, within 72 h of incubation at 70°C⁷ which helps the accessibility of the films to the enzyme. Being able to detect monomers within a few weeks of incubation at room temperature in a complex mixture, such as the plant digestive fluid, represents groundbreaking evidence of hydrolytic potential in such systems.

Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) analysis of partially hydrolyzed polyesters

Surface characterization of the after-treatment films via both FT-IR and SEM was used to bring further evidence of the incubation derived film erosion. The analysis was carried out for those polymers that were available after incubation. Most of the samples showed a gelatinous layer that was removed through extensive washing in urea prior to analysis. PET spectra of N.alata-incubated polymers are plotted in Fig. S2 (ESI). While there is almost complete superimposition of the original spectrum and only polymer condition spectrum, the normalized absorbance shifts progressively from the original profile in the incubated samples, especially PET/Tm_a/JA, in accordance with the HPLC analysis for this sample. The difference is visible in all the main peaks of the spectra, while it is mainly relevant in the 1710 cm^− 1, corresponding to aryl ester stretching. Other groups, whose decrease indicates the hydrolysis are 1240 cm^− 1, and 1090 cm^− 1, corresponding respectively to C = O carbonyl stretching, C-O stretching of ester group and vibrations of ester C-O bond. Similarly, changes in the PBAT surface upon incubation in N.alata pitcher fluids (Fig. S3) were seen such as a reduction of the peak at 1710 cm^− 1, mostly marked in PBAT/Tm_a/JA. Moreover, a decrease in relative absorbance was recorded at 1250 cm^− 1, 1160 cm^− 1, 1100 cm^− 1 area, as C = O stretching and CH plane bending.

Longitudinal sections of the plants (Fig. S4 in ESI) as well as the residual polymers already analyzed through FT-IR, were further characterized via SEM. Despite a similar extent of hydrolysis according to TPA release, for both polymers, SEM pictures (Figs. 3 and S5) indicated a different mechanism of hydrolysis for PBAT and PET. For PBAT, massive erosion with cavities was seen after 5 weeks of incubation, indicating vertical progressing of hydrolysis. On the other hand, for PET a more homogenous pattern was seen. Hydrolysis patterns are known to be not only polymer dependent. In fact, different types of polyester hydrolases show different erosion modes. As an example, lipases preferentially catalyze vertical hydrolysis, compared to cutinase from Fusarium solani⁴¹. Here, the most visible degradation profile was observed in PBAT/Tm_a (alive mealworm, no hormone). Figures S6, 7 (ESI) highlights the same conditions incubated in S. purpurea pitchers. Similar findings were acknowledged in Quartinello et al.⁶. Consistently with the data from the recovered monomers, the films were degraded to a minor extent.

Despite the fact that FT-IR and SEM provides a qualitative analysis, the results align with the previous HPLC derived consideration. Overall, all analysis results suggested more pronounced hydrolysis of polyesters in N. alata pitchers. Therefore, in the next step, studies towards the responsible proteins were conducted only for this plant.

Proteomics

Proteomic analyses were performed exclusively on N. alata pitcher fluids, as the HPLC results for S. purpurea fluids showed few significant differences between conditions and released far less TPA compared to Nepenthes. Additionally, the lack of available reference proteomes for Sarracenia species, further limited the feasibility of proteomic analysis for these samples.

Proteins were quantified through Bradford before and after the concentration through Vivaspin columns. Outputs are summarized in table S6.

In HeLa quality control standards, 3752 proteins were identified in line with the range reported in literature, 2700-6000^{42,43,44,45,46}. After filtering out contaminants and not consistent proteins across replicates, a total of 736 proteins were associated to Nepenthes pitcher fluids.

TPA release data indicated most efficient hydrolysis of polyesters in the presence of alive T. molitor (PET/Tm_a/JA; PET/Tm_a; PBAT/Tm_a; PBAT/Tm_a/JA). Therefore, these samples were compared to those containing dry T. molitor (PET/Tm_d, PBAT/Tm_d) at every time point (see Table S2 for a list of comparisons). Aspartic proteinase nepenthesin-1 (Uniprot-ID: Q766C3) was present in 83% of the comparisons and overabundant in 70%, while carboxypeptidase (Uniprot-ID: A0A140GML6) was present in 50% and overabundant in 63% thereof. When also including live mealworm conditions Tm_a and Tm_a/JA in the comparison, carboxypeptidase no longer met the criteria suggesting that its presence is influenced by the presence of mealworm rather than by the polyesters. However, after accounting for FDR by the Benjamini-Hochberg method, also p-values of nepenthesin rose from between 0.004 and 0.05 to values between 0.1 and 0.5. Hence, no significant influence of the different feed stimuli (alive or dry mealworm, JA addition or type of polyester) on the abundance of nepenthesin or other proteins was observed.

Molecular docking

The high abundance of nepenthesin in samples with elevated TPA release prompted further analysis. Alignment of amino acid sequences with well-known polyester hydrolyzing enzymes such as Leaf and Branch compost cutinase (LCC) and H. insolens cutinase (HiC) did not reveal any homology of nepenthesin (Table S3, ESI). Similarly, no structural similarity was observed when superimposing the 3D-structures.

For molecular docking, pepstatin, competitive aspartic protease inhibitor⁴⁷ was chosen as baseline due to its strong binding to nepenthesin’s active site. Pepsin (PDB: 1PSO) was chosen as reference (sequence identity with nepenthesin: 34.7%, E-value: 3∙10^− 11, RMSD: 1.788 Å for 186 aligned residues, indicating conserved active sites and high structural similarity (see Fig. S8)) to validate the docking procedure. Re-docking rigid pepstatin, co-crystallised with pepsin, to pepsin resulted in a binding affinity of -15.96 kcal∙mol^− 1, which was reduced by half (-7.91 kcal∙mol^− 1) when enabling ligand flexibility. Docking pepstatin to nepenthesin positioned the hydroxyl group of the statin residue in the third position of pepstatin in close proximity to the aspartic acid residues of the active site, which is consistent with the reported mechanism of inhibition⁴⁸ (Fig. S9). The binding affinity of pepstatin to nepenthesin was − 7.47 kcal∙mol^− 1. As an example of known effective hydrolysis activity, the polyesters PET and PBAT were docked to PHL7 resulting in binding affinities of -4.50 kcal∙mol^− 1 and − 4.75 kcal∙mol^− 1, respectively. The validity of the binding poses was confirmed by interactions previously described in literature^{39,49,50,51,52,53} including π-stacking of aromatic rings with F36 and W156, hydrophobic interactions of L210 and I179 and the proximity of the ester bond to S131 of the catalytic triad (Fig. S10).

The docking analysis of PET and PBAT to nepenthesin revealed several binding poses with ester bonds within 3 Å − 6 Å of catalytic aspartate residues (D35 and D237). These poses were stabilized by several hydrophobic interactions and hydrogen bonds. Residues F201 and Y244 on either end of the binding cleft were observed to engage in π-stacking with the aromatic rings of the polyesters (Fig. 4). Binding affinities were comparable to pepstatin, ranging from − 7.7 kcal∙mol^− 1 to -6.8 kcal∙mol^− 1 for PET and − 6.8 kcal∙mol^− 1 to -6.3 kcal∙mol^− 1 for PBAT. The lower binding affinities compared to PHL7 could be explained by the design of the active sites. PHL7’s active site is located on the surface of the enzyme, while nepenthesin’s long binding cleft is partially covered by a hairpin flap allowing more interactions with surrounding residues.

In aspartic proteases, the hydrolytic activity towards peptide bonds is mediated through a water molecule, activated by the active site aspartic acid residues⁵⁴. The close proximity of the ester bonds to the active site could make them susceptible to nucleophilic attack by such an activated water molecule, resulting in the hydrolysis of the polyesters. This theory is supported by a 2021 study of Bose and Zhao⁵⁵ who already demonstrated that a synthetic enzyme, mimicking the aspartic protease mechanism, was capable of hydrolysing the aromatic esters p-nitrophenyl acetate and p-nitrophenyl formate. Therefore, nepenthesin could indeed be responsible for hydrolysis of polyesters in N.alata pitcher fluid.

Hydrolysis of PET and PBAT by nepenthesin-1

Since the aspartic proteinase nepenthesin was highly abundant in conditions leading to better hydrolysis of polyesters in pitcher N. alata fluids, hydrolysis of the polyesters by recombinant nepenthesin-1 was investigated. Nepenthesin-1 from N.distillatoria was used in these experiments, as the enzyme from N. alata was not commercially available. Nevertheless, the nepenthesin identified in the proteomic study and the one from N.distillatoria share 43.7% sequence identity with 99% query coverage and an E-value of 9∙10^− 73, supporting its use as representative enzyme. Nepenthesin-1 showed an esterase activity on p-NPB of 794 U∙mg^− 1 and retained circa 20% of the activity after 72 h incubation at 37 °C (Table S7, ESI). Under these conditions, incubation of PET and PBAT with nepenthesin-1 lead to significant depolymerization (See HPLC chromatograms and histogram in Figs. S11–14), with TPA concentration of 0.28 mM TPA for PET and 0.72 mM TPA for PBAT. In comparison, the well-studied H. insolens cutinase⁷ incubated at the identical protein concentration yielded 0.21 mM TPA for PET and 0.28 mM for PBAT. No monomers were found in the controls (polymers with only water or only enzyme solution without polymers). The residual films were also washed (same protocol as for the preparation) and characterized in surface morphology through SEM. While PET surfaces showed limited signs of erosion (Fig. S15), PBAT exhibited a similar degradation pattern as seen after incubation in N. alata pitchers (Fig. S16, see also Fig. 2). These results find consistency with previous research on polyester hydrolysis by means of serine proteases^56,57 although at the best of the authors´ knowledge, not updated extensive characterizations of aspartic proteases activity on polyesters exist. Nepenthesin, in particular, has already established biotechnological application for mass spectrometry preparation, as an example⁹. Its protease activity regulation, as well as its specific cleavage site, have been measured⁵⁸ but at the moment, without any focus on polyesters as substrate. However, some works related nepenthesin proteolytic activity with the presence of insects (Drosophila melanogaster)⁵⁸ either because of indirect acidification of the pitcher fluid, which would activate the protease, or through signaling due to mechanical stimuli¹⁹. Among the highest TPA concentrations were in fact those samples incubated with hormone and mealworms, which additionally supports the mediated activation of pitcher enzymes.